Bioinformatics Analysis, Expression Profiling, and Functional Characterization of Heat Shock Proteins in Wolfi-poria cocos

Heat shock proteins (HSPs) play critical roles in regulating different mechanisms under high-temperature conditions. HSPs have been identified and well-studied in different plants. However, there is a lack of information about their genomic organization and roles in medicinal plants and fungi, especially in Wolfi-poria cocos (W. cocos). We identified sixteen heat shock proteins (HSPs) in W. cocos and analyzed in terms of phylogenetic analysis, gene structure, motif distribution patterns, physiochemical properties, and expression comparison in different strains. Based on phylogenetic analysis, HSPs were divided into five subgroups (WcHSP100, WcHSP90, WcHSP70, WcHSP60, and WcsHSP). Subgroups WcHSP100s, WcHSP90s, WcHSP70s, WcHSP60, and WcsHSPs were further divided into 3, 2, 3, 1, and 6 subfamilies, respectively. Moreover, the expression profiling of all HSP genes in five strains of W. cocos under different temperature extremes revealed that expression of most HSPs were induced by high temperature. However, every subfamily showed different expression suggesting distinctive role in heat stress tolerance. WcHSP70-4, WcHSP90-1, and WcHSP100-1 showed the highest response to high temperature stress. Heterologous expression of WcHSP70-4, WcHSP90-1, and WcHSP100-1 genes in Escherichia coli enhanced survival rate of E. coli during heat stress. These findings suggest the role of W. cocos heat shock genes in the high temperature stress tolerance.


Introduction
Heat shock proteins (HSPs) are newly synthesized and increased by organisms or cells within a certain period of time under high-temperature stress [1]. According to the size and structure of proteins, HSP family members are divided into five groups, namely HSP100, HSP90, HSP70, HSP60, and sHSP, involved in various aspects of cellular function. For example, most of the HSP100 proteins are ATP-dependent proteases with ATPase activity and molecular chaperone activity, which interact with other proteins and cooperate with other molecular chaperones to repair denaturing proteins and prevent faulty proteins from polymerizing [2,3]. The main function of HSP90 is the final maturation of proteins and the structural assembly of complex supramolecular proteins, and it also interacts with some kinases and transcription factors [4]. HSP70 mainly promotes protein degradation by binding to proteins to be degraded and localizing them to the proteasome and lysosome [5,6]. HSP60 consists of two subfamilies: one found in the cytoplasm of eukaryotes and archaea [7], and the other found in chloroplasts, mitochondria, and bacteria [8]. The primary function of HSP60 is the provision of assistance in prop folding of other proteins and prevention of aggregation and accumulation of misfolded conformation of proteins [9,10]. sHSPs protect organisms from abiotic stress damage by preventing irreversible protein aggregation and scavenging cellular reactive oxygen species. However, HSPs are synthesized in large quantities when an organism is subjected to high-temperature stress and act as molecular chaperones [11]. By binding to the reactive active region of proteins, they prevent misfolding or random aggregation of proteins by binding with active regions of proteins and lead the proteins to form an appropriate conformation. In this way, protein denaturation can be prevented and adverse effects caused by high-temperature stress on the growth and development of organisms can be alleviated, protein homeostasis can be maintained and cells can be protected from high-temperature damage [12].
In general, temperature plays an important role in the growth and development of fungi, including fungal growth, spore germination and reproduction [13]. For example, when the mycelium of Pleurotus eryngi is subjected to high-temperature stress, its growth rate decreases and the mycelium cells become short and thick [14]. Although Ganoderma lucidum is a large fungus that requires high temperature and good air for normal growth, when the temperature exceeds 35 • C, the growth of mycelium is inhibited and leads to problems such as thin cap and poor quality [15]. Several studies showed that G. clavigera is normally grown at lower temperatures, but O. montium grows at warmer temperatures [16][17][18]. In addition, high temperature affects both morphological structure and physiological activities of fungus, such as an increase in protein denaturation and reactive oxygen species (ROS) production with cell metabolism and related gene expression [19]. W. cocos, also called tuckahoe, grows on the roots of pines and is distributed in East Asia, India, and some American states [20]. The sclerotium of W. cocos is a precious source of traditional Chinese medicine with the roles in strengthening the spleen and heart soothing [21]. However, the mechanism of high temperature involved in affecting the growth rate and sporulation is still unknown in W. cocos. Furthermore, the production of HSPs is a kind of adaptation of life to the environment. In this study, therefore, we identified and characterized the HSP family genes from W. cocos, and determined the expression patterns of these genes under high temperature stress. Our results lay a foundation to understand the molecular mechanism of W. cocos HSPs in response to high temperature, and improve the production practices of W. cocos industry.

Experimental Materials and Heat Treatment
Five W. cocos strains, including 28 (ACCC 51641), 86 (ACCC 51642), 5.78 (ACCC 50864), 5.908 (CGMCC 5.908), and S10 (ACCC 51976), were obtained from the Chongqing Academy of Chinese Materia Medica, Chongqing, China. They were grown on Potato Dextrose Agar (PDA) medium at 28 • C and kept in the refrigerator at 4 • C for subsequent experiments. E. coli strains DH5α and BL21 (DE3) were provided by the Horticultural Plant Development and Stress Biology Innovation Team Laboratory, College of Horticulture and Landscaping, Southwest University.
All the five varieties of W. cocos were used in the heat treatment, and we inoculated the mycelian on PDA medium with a perforator. When the mycelian grew to 6 cm under the optimum temperature, they were placed in the incubator at different temperatures. The mycelial growth was measured by a vernier caliber. At least three biological repetitions were performed for each treatment. The method of multiple comparison of Least Significant Difference (LSD) was used to analyze the differences.
The same method was used in the high temperature treatment of mycelium, and the samples at 0 h, 1 h, 6 h, 12 h, 24 h and 48 h were collected; those collected at 0 h were used as the control. At least three biological repetitions were performed for each treatment and all samples were collected and frozen in liquid nitrogen immediately for RNA extraction.

W. cocos Sequence Analysis and Phylogenetic Tree Construction
The mycelia of W. cocos '28' were sent to Novogene Co., Ltd Beijing, China. for transcriptome sequencing, the coding sequence (CDS), and amino acid sequence of the corresponding heat shock protein-related genes were retrieved (Tables S1 and S2). Further, the Basic Local Alignment Search Tool (Blast) was performed and nucleotides with high homology and similar species were selected for further studies. The National Centre for Biotechnology Information (NCBI) conserved domain database (https://www.ncbi. nlm.nih.gov/Structure/cdd/cdd.shtml (accessed on 11 January 2022) was used to check the integrity and completeness of heat shock protein domain in the selected sequences. Phylogenetic trees were constructed and verified by way of the Neighbor-Joining (NJ) method using MEGA V6.0 with 1000 repeated guidance tests. The conserved domains of heat shock transcription factors and heat shock proteins of W. cocos. were analyzed by MEME (https://meme-suite.org/meme/tools/meme (accessed on 15 January 2022).

RNA Extraction and cDNA Synthesis
Total Ribonucleic Acid (RNA) was extracted from five kinds of W. cocos hyphae using the ominplant RNA Kit (DNaseI) Kit (Cwbio). The concentration and quality of RNA were verified by K5800-ultrafine spectrophotometer and 1.5% agarose gel electrophoresis, respectively. All obtained RNAs were retroscribed using DNaseI, RNASe-Free kits, and HiFiScript genomic deoxyribonucleic acid (gDNA) Removal complementary deoxyribonucleic acid (cDNA) Synthesis Kit (Cwbio) reverse transcription Kit. cDNA was stored in a −20 • C refrigerator for subsequent experiments.

Expression Analysis by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Gene-specific primers were designed using Primer Premier 7.0 (Table S3). The Qtower3G fluorescence quantitative Polymerase Chain Reaction instrument was used for qRT-PCR analysis. PCR conditions were the following: 95 • C for 10 min, 95 • C for 15 s, followed by 40 cycles of 59-63 • C (depending on the specific gene) for 30 s, and melting curve analysis were performed over a temperature range of 65-95 • C in 0.5 • C increments. The reaction mixture was 5 µL of 2 × UltraSYBR Mixture (Low ROX), 0.5 µL of each primer (10 µM), 2 µL of cDNA, and 2 µL of ddH 2 O. All cDNA samples were set with three technical replicates and a negative No Template Control (NTC) was set at the same time to analyze the relative expression levels of genes under the optimal annealing temperature. The amplification efficiency of each gene was within the 0.95-1.05 range, and R 2 was greater than 0.99. GADPH and ACT were used as an internal control and the relative expressions were calculated by the 2 −∆∆Ct method [22]. All data are means ± SD of three technical replicates; the 0 h sample was taken as control, two-way ANOVA was employed to analyze the data, and significant differences are represented by asterisks: * p < 0.05; ** p < 0.01.

Cloning of Heat Shock Protein Target Gene
According to the qRT-PCR results, the differentially expressed genes were selected for full-length specific amplification using Phanta ® Max super-fidelity DNA polymerase. The amplification conditions were as follows: 95 • C for 3 min, then 35 cycles of 95 • C for 15 s, 59 • C for 15 s, 72 • C for 2 min, and finally 72 • C for 5 min. The amplified products were verified and purified by 1.5% agarose gel and Universal DNA Purification Kit (Tiangen, Beijing, China), respectively. Specific primers were used for amplification (Table S2). The product was cloned into the pMD19-T vector (Southwest University, Chongqing, China). The gene was transformed into E. coli DH5α competent cells (Southwest University, Chongqing, China), and the positive clone plaque was identified by Taq enzyme using universal primer M13F along with gene-specific primers before and after the gene sequencing. Plasmid extraction was performed using the correctly sequenced bacteria through the FastPure Plasmid Mini Kit. Plasmid Extraction Kit was used to determine the Plasmid concentration.

Construction of Recombinant Plasmid Prokaryotic Expression Vector
The selected fragments of the target gene and the vector information of pET-32a (+) were analyzed and double digested with NotI and KpnI restriction sites. Primers for the target genes containing the restriction sites were designed using Primer Premier 7.0 ( Table S2). The purified plasmid was used as a template and the Phanta ® Max Super-Fidelity DNA was used to specifically amplify the full length of the restriction site. The amplified PCR products were detected by gel electrophoresis. The Universal DNA Purification Kit (Tiangen, Beijing, China) was used for DNA extraction and purification according to the manufacturer's guidelines. The ligation product was transferred into E. coli DH5α. Taq enzyme was used to identify the positive clone plaque by using the general primer S-tag and the back primer of the target gene and the general primer T7-TER and the forward primer of the target gene. Finally, the constructed recombinant vector plasmid and pET-32a (+) plasmid were introduced into the E. coli BL21.

Prokaryotic Expression and Heat Resistance
E. coli BL21 containing pET-32a (+) expression vector was inoculated in Luria-Bertani (LB) medium containing ampicillin (Amp) resistance at 1% volume fraction, expanded to optical density 600 (OD 600 ), 0.6-0.8 at 37 • C, induced by 1 mM Isopropyl-beta-Dthiogalactopyranoside (IPTG) for 3.5 h, and then adjusted to OD 600 , 0.6-0.8 again. The difference of each OD 600 was kept within 0.002. After induced expression, 200 µL of the bacterial solution was transferred to a centrifuge tube and was heated in a water bath at 50 • C for 0 min, 30 min, 60 min, 90 min, 120 min, and 150 min, respectively. A total of 2 µL of samples was extracted into LB medium plate and was cultured at 37 • C for 12-16 h, with 3 replicates per gradient.

The Heat-Resistant Analysis of Different W. cocos Strains
In order to analyze the resistance of different W. cocos strains to the high temperature, experiments of different temperature treatments were carried out in five strains of W. cocos. By measuring the speed of the mycelial growth shown in Figure 1, we determined that the '28' strain was the fastest to grow and the 'S10' was the one to do so the most slowly out of the five strains. However, the growth of strains mycelium could be inhibited by the high temperature ( Figure 1). Speed of growth, the density and color of mycelia ( Figure S1) showed that the majority of strains were influenced at 35 • C and severely inhibited at 38 • C, but different strains also had some differences. The '28' and '5.78' strains showed steady growth at 30-34 • C; despite the high temperature stress, the mycelium could maintain a certain growth speed at 35-37 • C. Endurance of 'S10' and '86' under high temperature is relatively worse than that of the other three strains. As the temperature increased, the speed of growth decreased, then mycelium began browning at 38 • C. Strain '10' already stopped growing at 39 • C. The heat resistance of five W. cocos strains was as follows: '28' = '5.78' > '5.908' > '86' > 'S10'. 908', and 'S10' of W. cocos under temperature treatment. All data are means ± SD of at least three technical replicates, and use the method of multiple comparison of LSD to analysis the differences, significant difference is represented by different letters a-f, 'cm/d' means centimeter per day.

Identification and Characterization of HSP Family Genes in W. cocos
In this study, a total of 16 WcHSPs were identified in W. cocos based on HMM profile and BLASTP analysis (Table S1). Among them, three, two, four, one, and six proteins belonged to HSP100, HSP90, HSP70, HSP60, and sHSP families, respectively. These HSP genes from W. cocos were defined as WcHSPs and listed in Table 1. As shown in Table 1, the amino acid residues, including WcHSP100s, WcHSP90s, and WcHSP70s, are varied from 128 to 287, 904 to 1043, 695 to 800, and 612 to 780, respectively, while the amino acid residues of WcHSP60s member was 456. Further, the isoelectric point ranged from 4.75 (WcHSP90-1) to 9.03 (WcsHSP-3). The fat solubility index of all members was more than 70 and WcsHSP-4 had the highest (110) fat solubility index. Members of WcHSP100 and WcsHSP were predicted in the nucleus, while WcHSP70 proteins were mainly distributed in the cytoplasm.

Phylogenetic Analysis of Heat Shock Protein Family Genes in W. cocos
To reveal the evolutionary relationships of WcHSP members, the NJ phylogenetic tree was constructed using WcHSP proteins from eleven different species, including W. cocos, Trametes pu-bescens, Grifola frondosa, Rhizoctonia Solani, Lentinula edodes, Saccharomyces cerevisiae, Hypsizygus marmoreus, Tricholoma Matsutake, Pyrrhoderma noxium, Ganoderma boninense and Ophiocordyceps sinensis, respectively. As shown in Figure 2, the 16 WcHSPs were classified into five groups, including three HSP100s, two HSP90s, four HSP70s, one HSP60 and six sHSPs, respectively. Based on phylogenetic analysis, the HSP90 and HSP100 family genes showed high similarity with each other. However, the HSP60 family genes were closely related with some sHSP family genes. In addition, these identified HSP family genes from W. cocos '28' were clustered into the same groups with those homologous genes from MD-104 SS10 (United States), indicating that the genetic link between the cultivars and confirming that the heat shock protein sequences were conserved in different W. cocos.

Conserved Motif Analysis of Heat Shock Protein in W. cocos
In addition, we determined that most of the genes showed similar motif patterns within the same subgroup but different among the families (Figure 3). For example, no conserved motif was identified in WcHSP90 and WcHSP60 family genes (Figure 3), hence the functional of these family genes are quite different from the other subgroups. All members of WcHSP70 have almost similar motif patterns with few exceptions, and three motifs (Motif 2, 3, and 4) were present in all four members. However, Motif 10 was present in all members except WcsHSP-3. In addition, three motifs (Motif 4, 7, and 8) were detected in all members of WcHSP100, while WcHSP100-2 contained one extra Motif 7. Moreover, WcsHSP-1, WcsHSP-2, WcsHSP-3, and WcsHSP-6 showed more similarity with each other compared to other members of WcsHSP subfamily. However, WcsHSP-4 and WcsHSP-5 were also very similar to each other. These conserved motifs may hold the key to understanding the way different heat shock proteins contribute to different biological functions.

Expression Patterns of Heat Shock Proteins at Different Temperatures in W. cocos
In order to analyze the function of HSPs in the high temperature and the response to high temperature stress, we tested the expression of HSPs in five strains following heat treatments for different timeframes using qRT-PCR.
The results of expression of HSP100 showed that the level of WcHSP100-1 expression was higher than that of WcHSP100-2 and WcHSP100-3, especially at 1 h, and had a transient expression effect (Figure 4). WcHSP100-1 expression was significantly induced after high temperature treatments and higher in all W. cocos strains after 1 h of treatment at varied temperatures. However, WcHSP100-1 showed the highest expression in W. cocos '5.78' and lowest in 'S10' after 1 h treatment of different high temperatures. The different strains '28', '86', '5.78', '5.908', and 'S10' of W. cocos are shown. All data are means ± SD of three technical replicates; the 0 h sample is taken as control, two-way ANOVA was employed to analyze the data, and significant differences are represented by asterisks: * p < 0.05; ** p < 0.01.
In addition, WcHSP100-2 showed less expression than WcHSP100-1 in all strains, and there was no significant change in expression within the same strain after treatments for various time points. Furthermore, when compared to other strains, gene expression in W. cocos 'S10' was lower at all-time points, reflecting it lower responsiveness to temperature changes. Moreover, the expression level of WcHSP100-3 was inhibited when the temperature was above 35 • C. The expression levels of the WcHSP100-3 gene in W. cocos '86', '5.908', and 'S10' strains were not significantly changed, but strain '5.78' displayed a significant change in expression compared to control. Initially, the expression of '5.78' was increased, but it decreased with extension in time period, implying that expression is influenced by temperature, but not significantly (Figure 4). In addition, the expression levels of these genes were relatively high in W. cocos '5.78'. Thus, combined with the heat-resistant analysis, we suggest that the WcHSP100-1 is more important in response to high temperature stress.
As shown in Figure 5, the expression profiles of the most WcHSP90s could be induced by the high temperature. Among them, the expression of WcHSP90-1 was increased in different cultivars, then decreased with the increase in temperature and time, but not significantly. The highest and the lowest expressions were noticed in '5.78' and 'S10' strains, respectively. Following 6 h of treatment at 38 • C, the highest expression level was observed, indicating that high temperature induces WcHSP90-1 gene expression. Moreover, high temperature has a more significant effect on WcHSP90-2, which peaked at 1 h after 38 • C treatment, especially in the strain '5.908' (Figure 5). In short, WcHSP90-1 and WcHSP90-2 showed similar expression patterns and displayed an increase in expression with increase in temperature. The different strains '28', '86', '5.78', '5.908', and 'S10' of W. cocos are shown. All data are means ± SD of three technical replicates; the 0 h sample is taken as control, two-way ANOVA was employed to analyze the data, and significant differences are represented by asterisks: * p < 0.05; ** p < 0.01.
All the WcHSP70 genes also showed an increase in expression with the increase in high temperature. WcHSP70-1 and WcHSP70-2 have the same trend of high-temperature response, which was upregulated and induced by high temperature, but the expression level was relatively low. These two genes showed higher expression levels in '28' strains at 38 • C and treatment with all temperatures in '5.78' strain, implying that the WcHSP70-1 and WcHSP70-2 genes are less sensitive to high temperature. On the other hand, WcHSP70-3 and WcHSP70-4 displayed a more obvious response to high temperature. WcHSP70-3 displayed highest expression in comparison to other WcHSP70 genes and gained maximum expression at 1 h of heat treatment. WcHSP70-4 expression was low at first, increased significantly after 1 h, and then decreased, indicating that this gene is transient and can respond quickly to high temperature. What is more, when the mycelium was under the heat stress, the WcHSP70-4 could keep continuous high expression in the heat-resistant strains '5.78', '28' and '5.908', but did not remain high during the late phase in heat-sensitive strain 'S10' (Figure 6). The different strains '28', '86', '5.78', '5.908', and 'S10' of W. cocos are shown. All data are means ± SD of three technical replicates; the 0 h sample is taken as control, two-way ANOVA was employed to analyze the data, and significant differences are represented by asterisks: * p < 0.05; ** p < 0.01.
The expression of WcHSP60 was also tested and showed results similar to those of WcHSP-70, which increased with the increase in temperature, but later on decreased with time and showed the highest expression at 1 h (Figure 7). The expression level of WcHSP60 was relatively higher in W. cocos '5.78'. The different strains '28', '86', '5.78', '5.908', and 'S10' of W. cocos are shown. All data are means ± SD of three technical replicates, take the 0h sample as control; two-way ANOVA was employed to analyze the data and significant differences are represented by asterisks: * p < 0.05; ** p < 0.01.
WcsHSPs were reacting most violently and rapidly under the high temperature; in most cases, their expression could increase hundredfold before and after the heat treatment. WcsHSP-1 showed the highest expression at 34 • C after 1 h of treatment, then gradually decreased with an increase in temperature, indicating lesser response to rising temperature. The expression level of WcsHSP-2 increased significantly after 1 h of treatment at 34 • C and 38 • C and subsequently decreased with extension of treatment time. WcsHSP-3 expression increased with increasing temperature (Figure 8) and peaked at 1 h and 6 h after treatment. More importantly, after 12 h at 38 • C, the expression level was also high, indicating good heat resistance potential. WcsHSP-4 showed modest expression compared to other genes but increased with rising temperature and time, indicating that it can withstand high temperatures for a longer duration. The expression of WcsHSP-5 was low and almost similar to that of the control. The expression of WcsHSP-6 was high at high temperatures and increased with the rising temperature, with a peak after 1 h following various temperatures. Then, it dropped dramatically, indicating a transitory reaction to the high temperature. In short, WcsHSP-1, WcsHSP-2, WcsHSP-3, and WcsHSP-6 had relatively high expression levels at 1 h while the expression of WcsHSP-4 and WcsHSP-5 was inhibited. Further, all the WcsHSP genes showed high expression levels in the '5.78' strain. The different strains '28', '86', '5.78', '5.908', and 'S10' of W. cocos are shown. All data are means ± SD of three technical replicates; the 0 h sample is taken as control, two-way ANOVA was employed to analyze the data, and significant differences are represented by asterisks: * p < 0.05; ** p < 0.01.

Prokaryotic Expression and Thermal Function Analysis of Heat Shock Protein of W. cocos
Based on heat treatment of mycelial and the qRT-PCR results, WcHSP70-4, WcHSP90-1 and WcHSP100-1 were suggested to play important roles in response to the heat stress. Therefore, we cloned these three genes from cDNA extracted from strain '28' for transformation in E. coli BL21. According to Figure 9, after 0 min, there was no change in the growth of E. coli. However, with the extension of heat shock time, E. coli showed a downward trend of growth, but the E. coli which expressed the WcHSP70-4, WcHSP90-1, or WcHSP100-1 grew better than the control. Especially after heat treatment for 150 min, the control group showed no growth, but the experimental group could grow slowly. Beyond this, we also observed that WcHSP100-1 could protect the E. coli against heat stress better. These findings imply that heat shock proteins play a critical role in high-temperature resistance and these heat shock proteins can help W. cocos to cope with the high temperature stress.

Discussion
High-temperature stress is an important environmental stress that affects the growth and development of fungi. Therefore, it is necessary to understand the mechanism by which fungi respond to high temperatures. Heat shock reactions in Saccharomyces cerevisiae have been thoroughly studied [23,24], but little is known about heat shock reactions in Basidiomycetes. Basidiomycetes are one of the largest divisions of fungus and contain many commercially important fungi, e.g., W. cocos. It is important due to higher amounts of bioactive secondary metabolites and it has attracted the attention of researchers.
The hyphae of different strains of W. cocos were treated with different temperatures, and mycelium had the best growth state at 28 • C (Figure 1 and S1), consistent with the most reported studies [25,26]. Although the mycelium could keep a stable growth rate at 30-34 • C, the density and color of hyphae point out it had been influenced by the high temperature ( Figure S1). When the temperature rose above 35 • C, the mycelia growth was severely inhibited and began to browning, then stopped growing at 40 • C. These results showed that several strains of W. cocos had similar stress response and that may be caused by the similar climatic range. In any case, there still were some differences in sensitivity to high temperature between different strains; '28' and '5.78' strains had a better heat-resistance.
Previous investigations indicate that the reaction to heat stress is connected with the expression of heat shock genes. As a result, there is a rising interest in employing heat shock proteins to understand the molecular basis of heat stress response [27][28][29]. Therefore, we cloned the different WcHSPs from the W. cocos '28' strains, and bioinformatics analysis were performed. Finally, we obtained three WcHSP100s, two WcHSP90s, four WcHSP70s, one WcHSP60 and six WcsHSPs based on the transcriptome sequencing data and cloned them. In the present study, most of HSP family genes, including WcHSP90s, WcHSP70s, and WcHSP60, had highly conserved between the W. cocos '28' and W. cocos MD104, whereas the WcHSP100 family genes are different between them (Table S1). In addition, we also found that the WcsHSP family genes (sHSP-1, sHSP-4, sHSP-5) showed highly conserved, but others display the significantly differences between the W. cocos '28' and W. cocos MD104.
The results indicate that these HSP family genes have functional differentiation among the different W. cocos strains.
HSPs are synthesized in large quantities when an organism is subjected to hightemperature stress and act as molecular chaperones to protect the functional protein and cell in various stresses responses. Proteome analysis of Asparagus bicarporus also determined that genes of the HSP70 family played an important role in resisting heat stress [30]. The studies also showed that high-temperature stress could induce the expression of HSP60 protein in P. pellucosa to resist the oxidative damage caused by high temperature [31]. In addition, Hsp17, Hsp22, Hsp70, and Hsp90 genes of 'Glucoderma lucidum' hyphae were expressed in different degrees to resist heat stress [32]. Different kinds of edible and medicinal fungi produce different heat shock proteins. Furthermore, we investigated the expression of 16 heat shock proteins in different strains under different temperature treatments by qRT-PCR, and all genes were regulated by high temperature. The expression of most heat shock proteins was increased under heat shock and small molecular heat shock protein family genes showed the highest response. For example, WcsHSP-2 had a much stronger response than the control, demonstrating that heat shock proteins are involved in W. cocos' heat shock response. This result is consistent with the expression of heat shock protein genes in other edible fungi [15,[33][34][35][36]. In addition, we also determined that WcHSP70-4, WcHSP90-1 and WcHSP100-1 had different expressions in heat-resistant and heat-sensitive varieties. These three genes might play a significant role and contribute to the difference in heat resistance of different varieties.
As research reported, HSPs expression is regulated by HSFs, which are central regulators of HSPs expression and participate in heat stress response [37]. Herein, we also found WcHSF3 could interact with WcHSP60, WcHSP70, and WcHSP90 ( Figure S2), and interaction among different WcHSPs suggests that these can regulate or inhibit expression of each other [38]. Candida albicans responds to temperature via Hsf1 and Hsp90 to orchestrate gene expression and chromatin architecture, thereby enabling thermal adaptation and virulence [39]. Furthermore, we also analyzed the expression of WcHSFs under heat treatments; the results are shown in Figure S3. Through the trend comparison of HSFs and HSPs expression, we determined that the expression of WcHSF3 had the similar change trend to WcHSP70-2, WcHSP60, WcHSP100-3 and opposite trend to WcHSP90-2 under different temperature treatments ( Figure S4). Therefore, we conjectured that WcHSF3 might be an activator of WcHSP70-2, WcHSP60, WcHSP100-3 and a repressor of WcHSP90-2, as well as a significant regulator in increasing W. cocos adaptation in high temperature. However, the regulatory relationship between WcHSFs and WcHSPs needs to be further verified by more experimental confirmations.
Finally, in order to better verify the function of the heat shock protein gene of W. cocos, we performed the corresponding high temperature resistance of WcHSP using heterologous expression in E. coli. The results showed that the survival rate of E. coli containing recombinant plasmid was significantly higher than that of the control group and the heterologous expression of W. cocos heat shock protein enhanced the tolerance of E. coli to high temperature, suggesting that the W. cocos has a role in heat shock high-temperature stress tolerance. Our findings are consistent with prior findings, which revealed that HmHSP70 and HSP60 proteins can improve E. coli heat tolerance by expressing them in Eugenia eentinus and Lentinus eentinus, respectively [40][41][42]. HmHSP70 heterologous expression in transgenic tobacco strains improved heat stress resistance. These findings suggest that the heterologous expression of the HmHSP70 gene in the mushroom will also enhance response to heat stress. Moreover, similar results were reported about the HSP100 gene [37,43]. In short, heterologous expression of heat shock proteins can enhance the tolerance of or-ganisms to high temperature, indicating that heat shock proteins play important roles in high-temperature stress.
In general, our findings provide insight into a better understanding of the functional characterization of W. cocos heat shock proteins, and lay the theoretical basis for studying the molecular mechanism of heat resistance in W. cocos. Importantly, WcHSP100-1, WcHSP90-1 and WcHSP70-4 were considered to be the key family genes for responding to the high temperature in W. cocos, but the function identification and regulation mechanism of WcHSPs and WcHSFs in heat treatment need further studies.